This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Oligosaccharides are biopolymers composed of multiple monosaccharide units connected through glycosidic bonds. Structural characterization of oligosaccharides requires not only determination of the identity and sequence of each monosaccharide unit, but also differentiation of possible branching, linkage, epimeric and anomeric isomers. It has been shown previously, in our lab and elsewhere, that electron capture dissociation (ECD) and hotECD (hECD) generally provide more extensive cross-ring cleavages than the conventional collisionally activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) methods. These cross-ring fragments are instrumental in determining the branching and linkage sites. In this study, metal-adducted permethylated linear and branched oligosaccharide ions were subject to ECD at several energies to investigate the effect of metal ion and electron energy on their fragmentation behaviors as well as the general applicability of ECD to isomer differentiation. Update for 2010-2011: Electron capture dissociation (ECD) behavior of oligosaccharides depends on both the electron energy and the type of charge carriers. Lithiated and alkali-earth metal adducted oligosaccharides fragmented effectively under both low and high energy conditions, whereas other alkali metal adducted oligosaccharides fragmented only under the hot-ECD conditions. Here, we carried out a systematic semi-quantitative study on model oligosaccharides to further investigate the effects of varying the electron energy and the charge carriers on the ECD behavior of oligosaccharides. The results of the energy-dependent ECD experiments indicate that, as the electron energy increases, the metal-adducted oligosaccharide ions can undergo three different fragmentation processes: ECD, which dominates when the electron energy is between 0.5 and 6 eV;hot-ECD, as characterized by formation of certain cross-ring cleavages, which occurs when the electron energy is between 6 and 16 eV;and EID, as characterized by the presence of doubly-charged fragment ions, which occurs when the electron energy is above 9 eV. Although the onsets of both the hot-ECD and EID processes were independent of the type of the charge carriers, smaller charge carriers frequently led to a higher degree of EID, whereas larger charge carriers tend to provide more diverse cross-ring cleavages covering a broader range of residues under hot-ECD condition. The potential of ECD for isomer differentiation is being further investigated by performing ECD at various electron energies towards the structural characterization of oligosaccharide linkage isomers. For example, although isomer differentiation between LNT and LNnT could not be achieved using low-energy ECD, hot ECD was able to generate diagnostic ions for each isomer: the presence of 1,3A2 and 2,5A2 fragments in the MS/MS spectra confirmed the 1[unreadable]3 linkage on the N-acetylglucosamine residue in LNT, and the 2,4A2 and 3,5A2 fragments confirmed the 1[unreadable]4 linkage on this residue in LNnT.